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Conservation plots and consensus sequences are shown at the bottom. Protein alignments were performed and represented using CLC-Bio sequence viewer [32]. Reference organisms: L. rhamnosus GG, L. casei ATCC 334, L. paracasei subsp. paracasei ATCC 25302, L. zeae (accession no. WP_010489923.1), L. buchneri CD034, L. plantarum WCFS1, L. helveticus R0052, L. delbrueckii subsp. lactis

DSM 20072, L. delbrueckii subsp. bulgaricus ATCC 11842, L. curvatus CRL 705, L. brevis ATCC 367, L. pentosus KCA1, L. coryniformis (ulaE, accession no. WP_010012151.1; xfp, WP_010012483.1). (ZIP 2 MB) References 1. Beresford TP, Fitzsimons NA, Brennan NL, BIBW2992 Cogan T: Recent advances in cheese microbiology. Int Dairy J 2001, 11:259–274.CrossRef 2. Sgarbi E, Lazzi C, Iacopino BMS202 concentration L, Bottesini C, Lambertini F, Sforza S, Gatti M: learn more Microbial origin of non proteolytic aminoacyl derivatives in long ripened cheeses. Food Microbiol 2013, 35:116–120.PubMedCrossRef 3. Cogan TM, Beresford TP, Steele J, Broadbent J, Shah NP, Ustunol Z: Invited review: advances in starter cultures and cultured foods. J Dairy Sci 2007, 90:4005–4021.PubMedCrossRef 4. Fox PF, McSweeney PLH:

Cheese: an overview. In Cheese: Chemistry, Physics and Microbiology. General Aspects. 3rd edition. Edited by: Fox PF, McSweeney PLH, Cogan TM, Guinee TP. London, UK: Elsevier; 2004:1–18.CrossRef 5. Settanni L, Moschetti G: Non-starter lactic acid bacteria used to improve cheese quality

and provide health benefits. Food Microbiol 2010, 27:691–697.PubMedCrossRef 6. de Dea Lindner J, Bernini V, de Lorentiis A, Pecorari A, Neviani E, Gatti M: Parmigiano Reggiano cheese: evolution of cultivable and total Lck lactic microflora and peptidase activities during manufacture and ripening. Dairy Sci Technol 2008, 88:511–523.CrossRef 7. Santarelli M, Bottari B, Lazzi C, Neviani E, Gatti M: Survey on the community and dynamics of lactic acid bacteria in Grana Padano cheese. Syst Appl Microbiol 2013, 36:593–600.PubMedCrossRef 8. Gatti M, de Dea Lindner J, de Lorentiis A, Bottari B, Santarelli M, Bernini V, Neviani E: Dynamics of whole and lysed bacterial cells during Parmigiano-Reggiano cheese production and ripening. Appl Environ Microbiol 2008, 74:6161–6167.PubMedCentralPubMedCrossRef 9. Neviani E, Bottari B, Lazzi C, Gatti M: New developments in the study of the microbiota of raw-milk, long-ripened cheeses by molecular methods: the case of Grana Padano and Parmigiano Reggiano. Front Microbiol 2013, 4:1–14.CrossRef 10. Neviani E, de Dea Lindner E, Bernini V, Gatti M: Recovery and differentiation of long ripened cheese microflora through a new cheese-based cultural medium. Food Microbiol 2009, 26:240–245.PubMedCrossRef 11. Bove CG, de Dea Lindner CG, Lazzi C, Gatti M, Neviani E: Evaluation of genetic polymorphism among Lactobacillus rhamnosus non-starter Parmigiano Reggiano cheese strains.

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Caused by Intensive Strength BLZ945 order exercise. Med Sci Sports Exerc 2011,43(8):1544–1551.PubMedCrossRef 31. Trombold JR, Barnes JN, Critchley L, Coyle EF: Ellagitannin Consumption Improves Strength Recovery 2–3 d after Eccentric Exercise. Med Sci Sports Exerc 2010,42(3):493–498.PubMedCrossRef 32. Udani K, Singh BB, Singh VJ, Sandoval E: BounceBack™ capsules for reduction of DOMS after eccentric exercise: a randomized, double-blind, placebo-controlled, crossover pilot study. J Int Soc Sports Nutr 2009, 6:14–18.PubMedCrossRef 33. Dunlap KL, Reynolds AJ, Duffy LK: Total antioxidant power in sled dogs supplemented with blueberries and the comparison of blood parameters see more associated with exercise. Comp Biochem Physiol A Mol Integr Physiol 2006,143(4):429–434.PubMedCrossRef

Nirogacestat mw 34. Kay CD, Holub BJ: The effect of wild blueberry (Vaccinium angustifolium) consumption on postprandial serum antioxidant status in human subjects. Br J Nutr 2002, 88:389–398.PubMedCrossRef 35. Lotito SB, Frei B: Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic Biol Med 2006,15(41):1727–46.CrossRef 36. Lyall KA, Hurst SM, Cooney J, Jensen D, Hurst RD, Lo K, Stevenson LM: Short-term blackcurrant extract consumption on exercise-induced check details oxidative stress and lipopolysaccharide-stimulated inflammatory responses. Am J Physiol Regul Integr Comp Physiol 2009,297(1):R70–81.PubMedCrossRef 37. Pedersen BK: Edward F. Adolph Distinguished Lecture: Muscle as an endocrine organ: IL-6 and other myokines. J Appl Physiol 2009, 107:1006–1014.PubMedCrossRef 38. Powers SK, Jackson MJ: Exercise-induced oxidative stress: cellular mechanisms and impact on muscle

force production. Physiol Rev 2008, 88:1243–1276.PubMedCrossRef 39. Steenberg A, Fischer CP, Keller C, Moller K, Pedersen BK: IL-6 enhances plasma IL-1ra, IL-10 and cortisol in humans. Am J Physiol Endocrinol Metab 2003, 285:E433-E437. 40. McAnulty LS, Nieman DC, Dumke CL, Shooter LA, Henson DA, Utter AC, Milne G, McAnulty SR: Effect of blueberry ingestion on natural killer cell counts, oxidative stress, and inflammation prior to and after 2.5 h of running. Appl Physiol Nutr Metab 2011,36(6):976–984.PubMedCrossRef 41. Theodorou AA, Nikolaidis MG, Paschalis VP, Koutsias S, Panayiotou GP, Fatouros IG, Koutedakis YK, Jamurtas AZ: No effect of antioxidant supplementation on muscle performance and blood redox status adaptations to eccentric training. Am J Clin Nut 2011, 93:1373–83.CrossRef 42. Gomez-Cabrera MC, Domenech E, Romagnoli M, Arduini A, Borras C, Pallardo FV, Sastre J, Viña J: Oral administration of vitamin C decreases muscle mitochondrial biogenesis and hampers training-induced adaptations in endurance performance.

pneumoniae infections. Therefore, differences among strains in the resistance to complement and/or to antimicrobial peptides Emricasan mediated killing may account for differences in virulence [11, 15, 39]. In addition, a wealth of evidence clearly indicates the importance of the inflammatory responses in clearing K. pneumoniae infection and have provided substantial evidence for the protective role of a Th1-mediated response [40–42]. Thus, differences in the

induction of inflammatory responses among strains may also underline in vivo eFT508 molecular weight behavior. In summary the available data support the notion that CPS-dependent cytotoxicity, together with other bacterially triggered events, is required for virulence. Further studies will attempt to elucidate SC79 these novel virulence mechanisms, which may differ among capsulated strains, in order to achieve a comprehensive understanding of K. pneumoniae pathogenesis. Conclusion This study allocates a novel role to K. pneumoniae capsule, i.e. the induction of cytotoxicity during the infection of lung epithelial cells. This effect, which has been analysed

by using four different approaches, is not capsule serotype dependent, does require the presence of live bacteria, and does not seem to be directly related to bacterial adhesion. Host cell cytotoxicity could be associated with virulence. However, strains expressing different capsule levels were not equally virulent, suggesting that additional bacterial elements could be involved in Klebsiella virulence. Acknowledgements Salary support to V.C. from Govern Balear is gratefully acknowledged.

J.G. is a recipient of a Contrato de Investigador “”Miguel Servet”" from Instituto de Salud Carlos III. This work has been funded by grants from FIS (CP05/00027 to J.G. and PI06/1629 to J.A.B.). Ciberes is an initiative from Instituto de Salud Carlos III, Spain. The authors sincerely thank Dr. Christian selleck chemical Frank for critical reading of the manuscript. References 1. Carpenter JL:Klebsiella pulmonary infections: occurrence at one medical center and review. Rev Infect Dis 1990, 12:672–682.PubMed 2. Gupta A: Hospital-acquired infections in the neonatal intensive care unit- Klebsiella pneumoniae. Semin Perinatol 2002, 26:340–345.CrossRefPubMed 3. Jarvis WR, Munn VP, Highsmith AK, Culver DH, Hughes JM: The epidemiology of nosocomial infections caused by Klebsiella pneumoniae. Infect Control 1985, 6:68–74.PubMed 4. Bartlett JG, O’Keefe P, Tally FP, Louie TJ, Gorbach SL: Bacteriology of hospital-acquired pneumonia. Arch Intern Med 1986, 146:868–871.CrossRefPubMed 5. Straus DC: Production of an extracellular toxic complex by various strains of Klebsiella pneumoniae. Infect Immun 1987, 55:44–48.PubMed 6. Strauss E: A symphony of bacterial voices [news]. Science 1999, 284:1302–1304.CrossRefPubMed 7.

The experiment was repeated twice. Assays for sensitivity to antibiotics, detergents, and osmotic stress The sensitivity of R. check details leguminosarum bv. trifolii strains to sodium deoxycholate (DOC), sodium dodecyl sulfate (SDS), and ethanol was studied, and minimal inhibitory concentration of particular agents was determined. Bacteria were collected from TY agar medium into sterile water to an OD600 of 0.3 and 10 μl of each suspension was plated on TY containing a defined concentration of DOC (0.005-1% w/v), SDS (0.005-1% w/v) or ethanol (0.25-6% v/v).

After 3 days, the growth of strains on individual media was determined. Three independent experiments were done for each strain. To assess the effect of osmolarity on growth of the R. leguminosarum bv. trifolii Rt24.2 and the rosR mutants, Selleckchem Temsirolimus the strains were grown in TY medium supplemented with a defined concentration of NaCl (0-510 mM). Cultures were incubated at 28°C for 48 h, and then the OD600 was measured. Tolerance to hypo-osmotic stress was determined using low-osmolarity JNJ-26481585 glutamate-yeast extract-mannitol (GYM) medium

[35]. Antibiotic sensitivity assays were performed using commercially available filter disks with the appropriate antibiotic: ampicillin (10 μg), erythromycin (15 μg), chloramphenicol (30 μg), gentamicin (10 μg), bacitracin (10 μg), augmentin (30 μg), streptomycin (10 μg), polymyxin B (10 μg), carbenicillin (20 μg), penicillin G (10 U), and tetracycline (30 μg) (Mast Diagnostics, Merseyside, UK). Filter disks were placed on the surface of 79CA medium, where 100 μl of R. leguminosarum cultures were previously spread. The diameter of the growth inhibition zone was measured after 3 days of incubation. Isolation and analysis of extracellular and membrane proteins For analysis of extracellular and membrane proteins, the Rt2472 and Rt24.2 strains were grown at 28°C for 2 days to an OD600 of 0.6 in 200 ml TY medium. To study the influence of clover root exudates on membrane protein profiles, these strains were grown at 28°C for 3 days in 400 ml M1 medium supplemented 4��8C with Dilworth’s

vitamins and with or without 5 μM exudates. Cells were removed by twice centrifugation at 5,000 × g for 20 min at 4°C, and supernatants were used for purification of extracellular proteins. The proteins were concentrated by precipitation with 10% trichloroacetic acid according to the procedure by Russo et al. [14]. Membrane proteins from cell pellets were isolated according to the method described by Kucharczyk et al. [70]. The cells were washed in 200 ml 50 mM Tris-HCl (pH 7.4), and centrifuged at 5,000 × g for 20 min at 4°C. Cell pellet was resuspended in 1.6 ml 200 mM Tris-HCl (pH 8.0), and then 1.6 ml 1 M sucrose in 200 mM Tris-HCl (pH 8.0), 16 μl lysozyme (12 mg/ml in 100 mM EDTA, pH 8.0) and 3.2 ml ice cold water were added. Next, 25.6 μl saturated ethanol-phenylmethylsulfonylfluoride (PMSF) solution and 12.

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Wijnen AJ, Lian JB, Montecino M, Croce CM, Choi JY, Ali SA, Pande S, Hassan MQ, et al.: Transcription factor-mediated epigenetic regulation of cell growth and phenotype for biological control and cancer. Adv Enzyme Regul 50: 160–167. 11. Kajanne R, Miettinen P, Tenhunen M, Leppa S: Transcription factor AP-1 promotes growth and radioresistance in prostate cancer cells. Int J Oncol 2009, 35: 1175–1182.PubMed 12. Song Y, Wu J, Oyesanya RA, Lee Z, Mukherjee A, Fang X: Sp-1 and c-Myc mediate lysophosphatidic acid-induced expression of vascular endothelial growth factor in ovarian cancer cells via a hypoxia-inducible factor-1-independent mechanism. Clin Cancer Res 2009, 15: 492–501.PubMedCrossRef 13. Blyth K, Cameron ER, Neil JC: The RUNX genes: gain or loss of function in cancer. Nat Rev Cancer 2005, 5: 376–387.PubMedCrossRef 14. Li Y, Tian B, Yang J, Zhao L, Wu X, Ye SL, Liu YK, Tang ZY: Stepwise metastatic human hepatocellular see more carcinoma cell model system with multiple metastatic potentials established through consecutive in vivo selection and studies on metastatic

Dapagliflozin characteristics. J Cancer Res Clin Oncol 2004, 130: 460–468.PubMedCrossRef 15. Deregibus MC, Cantaluppi V, Doublier S, Brizzi MF, Deambrosis I, Albini A, Camussi G: HIV-1-Tat protein activates phosphatidylinositol 3-kinase/AKT-dependent survival pathways in Kaposi’s sarcoma cells. J Biol Chem 2002, 277: 25195–25202.PubMedCrossRef 16. Hijiya N,

Setoguchi M, Matsuura K, Higuchi Y, Akizuki S, Yamamoto S: Cloning and characterization of the human osteopontin gene and its promoter. Biochem J 1994, 303 (Pt 1) : 255–262.PubMed 17. Shevde LA, Das S, Clark DW, Samant RS: Osteopontin: An Effector and an Effect of Tumor Metastasis. Curr Mol Med 2010, 10 (1) : 71–81.PubMedCrossRef 18. Johnston NI, Gunasekharan VK, Ravindranath A, O’Connell C, Johnston PG, El-Tanani MK: Osteopontin as a target for cancer therapy. Front Biosci 2008, 13: 4361–4372.PubMedCrossRef 19. Jain S, Chakraborty G, Bulbule A, Kaur R, Kundu GC: Osteopontin: an emerging therapeutic target for anticancer therapy. Expert Opin Ther Targets 2007, 11: 81–90.PubMedCrossRef 20. Wai PY, Kuo PC: Osteopontin: regulation in tumor metastasis. Cancer Metastasis Rev 2008, 27: 103–118.PubMedCrossRef 21. Schultz J, Lorenz P, Ibrahim SM, Kundt G, Gross G, Kunz M: The functional -443T/C osteopontin promoter polymorphism influences osteopontin gene expression in melanoma cells via binding of c-Myb transcription factor. Mol Carcinog 2009, 48: 14–23.PubMedCrossRef 22. Ramsay RG, Gonda TJ: MYB function in normal and cancer cells.

No full-length EscU (39 kDa) was detected in the ΔescU/pJLT24 membrane fraction, suggesting complete auto-cleavage

had occurred under these conditions. EscU(N262A) was detected exclusively at 39 kDa with anti-HA antibodies. Interestingly, EscU(P263A) appeared as a 39 kDa polypeptide along with a 29 kDa and 10 kDa polypeptides detected by anti-HA antibodies and NCT-501 anti-FLAG antibodies respectively. These data demonstrate that the EscU 29 and10 kDa auto-cleavage products localized to membrane fractions enriched for T3SS selleck chemicals needle complexes and are in agreement with the crystal structure soluble domain interactions previously reported [26]. In addition, plasmid encoded EscU(P263A) is auto-cleaved in EPEC albeit at reduced levels compared to normal EscU. Figure 2 EscU auto-cleavage results in a 10 kDa C-terminal product that is membrane associated in EPEC. (A) Isolated membrane fractions were probed with anti-HA

and anti-FLAG antibodies to assess EscU auto-cleavage status. Membrane localization of EscJ is unchanged AZD1480 in escU null mutants (lane 2) and therefore this protein served as an internal control for the individual membrane fractions. The approximate 10 kDa C-terminal EscU auto-cleavage product (detected with anti-FLAG antibodies) along with the 29 kDa HA-tagged N-terminal product (detected with anti-HA antibodies) both partitioned to the membrane fraction (denoted by arrows). Uncleaved EscU is also membrane associated and appeared as a 39 kDa species. (B) The same membrane fractions were probed with anti-EscN antibodies to detect membrane associated EscN levels. A ΔescN mutant membrane preparation was included to demonstrate

the specificity of the antibody. The formation of functional T3SS needle complexes is believed to be a multistep process. For EPEC, T3SS needle complexes are less well characterized than those of Salmonella and Shigella species. Purified EPEC T3SS needle complex preparations often lack certain protein components that are highly conserved in all systems Florfenicol and hence expected to be part of a ‘complete’ T3SS needle complex. For example EscF, the putative needle protein has not been detected in highly purified EPEC needle preparations [20]. Antibodies to EscJ and EscN [39] were used to probe membrane fractions to assess the expression levels of these proteins. No change in the amount of cell envelope associated EscJ or EscN was observed in ΔescU bacteria expressing any of the EscU variants (Figure 2A and 2B). These data indicate that EscU auto-cleavage is not essential for EscN and EscJ localization to the cell envelope.

In the 3-deazaneplanocin A other two categories, score 0 and 3+, the agreement was substantial /almost perfect (greater than 0.80). Table 3 k cs statistic and 95% Jackknife confidence interval by HER2 score Score N slides kcs 95% Confidence

interval of kcs       Lower limit Upper limit 0 64 0.80 0.64 0.97 1+ 64 0.54 0.31 0.78 2+ 64 0.37 0.07 0.70 3+ 64 0.85 0.70 1.00 EQA HER2 interpretation Table 4 summarizes the results obtained from the EQA HER2 interpretation step. Only two PCs provided scores equal to reference ones for all the 10 slides. Four PCs provided one discordant value out of 10, misclassifying the reference value score 1+ in three cases and score 2+ in one case. It is worthy to note, that

no score 3+ was misclassified and only 1 score 0 was interpreted as score 1+. Conversely, we observed 12 and 14 misclassifications in score 1+ and 2+, respectively. Table 4 HER2 interpretation: misclassifications in relation to the reference score ID Group Total N° of misclassified slides(#) Reference score 0(#) Reference score 1 + (#) Reference BIBW2992 score 2 + (#) Reference score 3 + (#) PC1 3 1/10 0/2 1/3 [2+] 0/3 0/2 PC2 3 2/10 0/2 0/3 2/3 [1+;1+] 0/2 PC3 1 1/10 0/2 1/3 (*) 0/3 0/2 PC4 1 2/10 0/2 0/3 2/3 [1+;1+] 0/2 PC5 3 0/10 0/2 0/3 0/3 0/2 PC6 2 2/10 0/2 1/3 [2+] 1/3 [3+] 0/2 PC7 3 0/10 0/2 0/3 0/3 0/2 PC8 1 2/10 1/2 [1+] ^ 0/3 1/3 Thymidine kinase [1+] 0/2 PC9 2 1/10 0/2 1/3 [2+] 0/3 0/2 PC10 2 2/10 0/2 1/3 [2+] 1/3 [1+] 0/2 PC11 2 2/10 0/2 1/3

[2+] 1/3 [1+] 0/2 PC12 1 2/10 0/2 1/3 [2+] 1/3 [1+] 0/2 PC13 3 3/10 0/2 2/3 [2+;2+] 1/3 [1+] 0/2 PC14 1 1/10 0/2 0/3 1/3 [1+] 0/2 PC15 2 2/10 0/2 1/3 [2+] 1/3 [3+] 0/2 PC16 3 4/10 0/2 2/3 [0;0] 2/3 [1+;1+] 0/2 Total 27/160 1/32 12/48 14/48 0/32   (*) Slide not evaluated. (#)N° of misclassified slides/N° of received slides. ^Brackets report the score provided by PCs. Table 5 shows the kw values and the relative lower limit of the 95% confidence interval obtained by comparing the scores provided by PCs with the reference values. Overall, by considering the point-estimate values of the kw statistic a satisfactory agreement was reached between the reference score and the one provided by the evaluation of each PC. However, by taking into account the lower PLX4032 limits of the 95% confidence interval of the kw statistic, only 6 PCs reach a fully satisfactory agreement. Table 5 k w statistic values between the reference scores and scores reported by PCs ID Group kw kw 95% CI       Lower limit Upper limit PC1* 3 0.95 0.86 1.00 PC2 3 0.90 0.76 1.00 PC3* 1 1.00 1.00 1.00 PC4 1 0.90 0.76 1.00 PC5* 3 1.00 1.00 1.00 PC6 2 0.91 0.80 1.00 PC7* 3 1.00 1.00 1.00 PC8 1 0.89 0.72 1.00 PC9* 2 0.95 0.86 1.00 PC10 2 0.90 0.77 1.

) to serve as controls. Eppendorfs were inoculated with known saturating 3H-Leu (80 nM final concentration, specific activity: 73 Ci.mmol-1) and incubated in the dark for 2 h. Protein synthesis was stopped by the addition of formaldehyde SBI-0206965 mw (1.6% final concentration). Samples were then filtered through a 25-mm diameter, 0.22-μm pore size membrane (GTTP). The filters were then rinsed twice with 5 ml of trichloroacetic acid (TCA, 5% final concentration). The filters were placed in scintillation vials, allowed to dry and

solubilised with 1 ml of toluene. After adding 3 ml of the scintillation cocktail (Hionic Fluor, Perkin Elmer), the radioactivity was counted with a Packard Tricarb Liquid Scintillation Analyser 1500. Bacterial production, calculated in pmoles l-1 h-1 of 3H-Leucine incorporated into protein, was converted in μgC l-1 h-1 following Simon and Azam [62]: BP (μgC l-1 h-1) = Leu (mmols Leu L-1 h-1) × 131.2 × (%Leu)-1 × (C:Protein) × ID); with C:protein = 0.86 (ratio of cellular carbon to protein); %Leu = 0.073 (fraction of leucine in protein). ID = 1 (Isotopic Dilution); 131.2 = Molecular weight of the leucine. Estimation of viral production We used the dilution technique of Wilhelm et al. [63] in order to estimate the viral production BTSA1 throughout the experiment www.selleckchem.com/products/Rapamycin.html at day 0, 2 and 4. 50 ml of sub-samples were diluted and mixed with 100 ml of virus-free (0.02-μm pore size pre-filtered at day 0 and kept at 4°C) lake water, and

incubated in dark conditions. Triplicates were made and incubated at in situ temperature in the dark. One-ml sub-samples were collected at 0, 3, 6, 12, 18 and 24 h. Viral production rates were determined from first-order regressions of viral abundance versus time after correcting

for the dilution of the bacterial hosts between the samples and the natural community, a necessary step to account for the loss of potentially infected cells during the filtration. Viral production (VP, virus ml-1 h-1) was calculated as proposed by Hewson and Fuhrman [64]: VP = m × (b/B) where m is the slope of the regression line, b the 3-mercaptopyruvate sulfurtransferase concentration of bacteria after dilution, and B the concentration of bacteria prior to dilution. We estimated the number of lysed bacteria (cell ml-1 h-1) during the viral lysis activity by considering an average burst size (27) previously estimated for Lake Bourget [7, 65] with the number of lysed bacteria = Viral production/Burst Size [66]. In order to show the effect of the presence of flagellates on the dynamics and activities of both heterotrophic bacteria and viruses, we calculated the stimulation of the different parameters presented above (both abundance and production) in treatments VF and VFA (as proposed by Bonilla-Findji et al. [18] and Zhang et al. [22]). The stimulation corresponds to the difference in variation between treatments with flagellates (VFA or VF treatments) and the treatment without flagellates (V treatment) between 0 and 48 h, and between 48 h and 96 h, respectively.

The experimental platform was composed of submerged enclosures (1.2 m diameter and 2 m depth) which allowed the isolation of up to 2,000 L and the simulation of

UVBR and temperature increases in order to study the responses of pelagic communities to these manipulated factors simultaneously. The regulations of UVBR and temperature are performed with high frequency monitoring following the in situ temperature and natural incident UVBR (see details in supplementary data; full description in Bucladesine Nouguier et al. [25]). Four enclosures, filled with lagoon surface-water at random, were used as incubators for the 2 L experimental bags (UV-permeable sterile Whirl Pack® polyethylene bags incubated at subsurface) in which microbial communities were isolated. The factorial experimental

design constituted eight different treatments Caspase Inhibitor VI price (each being tested in three replicates): C: control, C + Nut: control with nutrient addition, UV: UVBR increase (+20%), UV + Nut: UVBR increase (+20%) and nutrient addition, T: temperature increase check details (+3°C), T + Nut: temperature increase (+3°C) and nutrient addition, TUV: temperature (+3°C) and UVBR (+20%) increases, TUV + Nut: temperature (+3°C) and UVBR increases (+20%) and nutrient addition (Figure 1). Figure 1 Crossed factorial experimental design conducted to assess the effects of the three regulatory factors: (Temperature, UVB radiation and nutrient increases). In order to fill the 24 Whirl Pack bags, 100 L subsurface lagoon water was pumped and pre-filtered through 6-μm-pore-size

polycarbonate membranes (47 mm in diameter) in order to isolate the smallest planktonic fraction. This water sample (<6 μm) was equally distributed into 24 sterile Whirl Pack® polyethylene bags. 12 of these experimental bags received nutrients addition at time zero, while the others were kept without nutrient addition. The set bags which represented the enriched Fludarabine order nutrient conditions were obtained by addition of a mixture of leucine (C and N) and phosphate in order to maintain a substrate C:N:P molar ratio close to that of marine bacteria [26] as described in Bouvy et al.[24]. The bags with and without nutrient addition exhibited concentrations of 0.20 μM and 0.07 μM of PO4, respectively. The two levels of P concentration mimicked natural fluctuations in coastal lagoon waters. These concentrations were chosen to be relevant to phosphorus concentrations recently measured in Thau lagoon (a general decrease over the past 30 years has led to low values of soluble reactive phosphorus: i.e. from 3 μM to undetectable values (<0.03 μM in winter) [27]). Since nutrients usually refer to inorganic nutrients, it should be noted that in this study, “nutrients” actually refer to “nutrients and organic source of C and N”.